CHAPTER 1 INTRODUCTON. Histopathology- Definition it is a branch of pathology which deals with the

Transcription

1 CHAPTER 1 INTRODUCTON Histopathology- Definition it is a branch of pathology which deals with the study of disease in a tissue section. The tissue undergoes a series of steps before it reaches the examiners desk to be thoroughly examined microscopically to arrive at a particular diagnosis. To achieve this it is important that the tissue must be prepared in such a manner that it is sufficiently thick or thin to be examined microscopically and all the structures in a tissue may be differentiated. The objective of the subsequent discussions will be to acquaint the staff with their responsibility; the basic details of tissue handling, processing and staining. The term histochemistry means study of chemical nature of the tissue components by histological methods. The cell is the single structural unit of all tissues. The study of cell is called cytology. A tissue is a group of cells specialized and differentiated to perform a specialized function. Collection of different type of cells forms an organ. Type of material obtained in laboratory The human tissue comes from the surgery and the autopsy room from surgery two types of tissue are obtained. 1

2 1. As biopsy- A small piece of lesions or tumor which in sent for diagnosis before final removal of the lesion or the tumor (Incisional biopsy). 2. If the whole of the tumor or lesion is sent for examination and diagnosis by the pathologist, it is called excisional biopsy. 3. Tissues from the autopsy are sent for the study of disease and its course, for the advancement of medicine. Types of Histological preparation The histological specimen can be prepared as 1. Whole mount 2. Sections 3. Smears. 1. Whole mounts- These are preparation entire animal eg. fungus, parasite. These preparations should be no more than mm in thickness. 2. Sections- The majority of the preparations in histology are sections. The tissue is cut in about 3-5 mm thick pieces processed and 5 microns thick sections are cut on a microtome. These are then stained and permanently mounted. Microtomes are special instruments which have automatic mechanism for cutting very thin sections. To cut the sections on the microtome; the tissue must be made hard enough to not get crushed. There are 2 methods of hardening the tissues. One is by freezing them and the other is by embedding them in a hard material such at paraffin wax or gelatin. 2

3 3. Smears- Smears are made from blood, bone marrow or any fluid such as pleural or ascitic fluid. These are immediately fixed in alcohol to presence the cellular structures are then stained. Smears are also made by crushing soft tissue between two slides or an impression smear in made by pressing a clean slide in contact with the moist surface of a tissue. By doing this, the cells are imprinted on the slide and these may be stained for cytological examination. 3

4 Responsibility of a technician The technician is responsible for 1. Specimen preservation. 2. Specimen labeling, logging and identification. 3. Preparation of the specimen to facilitate their gross and microscopy. 4. Record keeping. To obtain these aims the following point need consideration. 1. As soon as the specimen is received in the laboratory, check if the specimen is properly labeled with the name, age, Hospital Registration No. and the nature of tissue to be examined and the requisition form is also duly filled. 2. Also check if the specimen is in proper fixative. Fixative should be fifteen to twenty times the volume of the specimen add fixative if not present in sufficient amount. 3. Check if the financial matters have been taken care off. 4. Make the entries in biopsy register and give the specimen a pathology number called the accession number. Note this number carefully on the requisition form as well as the container. This number will accompany the specimen every where. 5. If the specimen is large inform the pathologist who will make cut in the specimen so that proper fixation is done. Container should be appropriate to hold the specimen without distorting it. 4

5 6. Blocks of tissues taken for processing should be left in 10% formalin at 60 C till processing. These would be fixed in 2 hours. 7. Slides should be released for recording after consultation with the pathologist. 8. Specimens should be kept in their marked container and discarded after checking with pathologist. 9. Block must be stored at their proper number the same day. Note the blocks have to be kept preserved for life long. Slides should be stored in their proper number after 3 days. It gives time for the slides to be properly dried. 5

6 CHAPTER -2 FIXATION Definition It is a complex series of chemical events which brings about changes in the various chemical constituents of cell like hardening, however the cell morphology and structural detail is preserved. Unless a tissue is fixed soon after the removal from the body it will undergo degenerative changes due to autolysis and putrefaction so that the morphology of the individual cell will be lost. Mode of teaching - Overhead projector and practical demonstration. Principle of fixation- The fixative brings about crosslinking of proteins which produces denaturation or coagulation of proteins so that the semifluid state is converted into semisolid state; so that it maintains everything in vivo in relation to each other. Thus semisolid state facilitate easy manipulation of tissue. Aims and Effects of fixation If a fresh tissue in kept as such at room, temperature it will become liquefied with a foul odour mainly due to action of bacteria i.e. putrefaction and autolysis so the first and fore most aim of fixation is 1. To preserve the tissue in as lf like manner as possible. 2. To prevent postmortem changes like autolysis and putrefaction. Autolysis is the lysis or dissolution of cells by enzymatic action probably as a result of rupture of lysosomes. Putrefaction The breakdown of tissue by bacterial action often with 6

7 formation of gas. 3. Preservation of chemical compounds and microanatomic constituents so that further histochemistry is possible. 4. Hardening : the hardening effect of fixatives allows easy manipulation of soft tissue like brain, intestines etc. 5. Solidification: Converts the normal semifluid consistency of cells (gel) to an irreversible semisolid consistency (solid). 6. Optical differentiation - it alters to varying degrees the refractive indices of the various components of cells and tissues so that unstained components are more easily visualized than when unfixed. 7. Effects of staining - certain fixatives like formaldehyde intensifies the staining character of tissue especially with haematoxylin. Properties of fixatives 1. Coagulation and precipitation as described above. 2. Penetration Fixation is done by immersing the tissue in fluid containing the fixative. Faster a fixative can penetrate the tissue better it is penetration power depends upon the molecular weight e.g. formalin fixes faster than osimic acid. 3. Solubility of fixatives - All fixatives should be soluble in a suitable solvent, preferably in water so that adequate concentrations can be prepared. 4. Concentration - It is important that the concentration of fixative is isotonic or hypotonic 7

8 5. Reaction - Most fixatives are acidic. It may help in fixation but can affect staining so has to be neutralized e.g. formalin is neutralized by adding of calcium carbonate. Amount of fixative The fixative should be atleast times the bulk of tissue. For museum specimens the volume of fixative is > 50 times. Note : If the specimen is large then see that the sections are made to make slices which have a thickness of 1.5 cm so that fixative can penetrate the tissue easily Reagents employed as fixatives (simple fixatives) I. Formaldehyde - Formaldehyde is a gas but is soluble in water to the extent of 37-40% w/v. This solution of formaldehyde in water is called formalin or full strength formalin. Formalin is one of the commonly used fixative in all laboratories since it is cheap penetrates rapidly and does not over harden the tissues. It preserves the proteins by forming crosslinkage with them and the tissue component. It denatures the proteins. Glycogen is partially preserved hence formalin is not a fixative choice for carbohydrates. Some enzymes can be demonstrated in formalin fixed tissues. It neither preserves nor destroys fat. Complex lipids are fixed but has 8

9 no effect on neutral fat. After formalin fixation fat may be demonstrated in frozen section. Pure formalin is not a satisfactory fixative as it overhardens the tissue. A 10% dilution in water (tap or distilled) is satisfactory. Since it oxidizes to formic acid if kept standing for long period so it should be neutralized by phosphates or calcium carbonate otherwise it tends to form artifact; a brown pigment in tissues. To remove this pigment picric alcohol or saturated alcoholic sodium hydroxide may be used. Concentrated formalin should never be neutralized as there is a great danger of explosion. The commercial formalin becomes cloudy on standing especially when stored in a cool place due to formation of precipitate of paraformaldehyde which can be filtered. Formalin on prolonged exposure can cause either dermatitis its vapour may damage the nasal mucosa and cause sinusitis. Time required for fixation. At room temperature - 12 hours For small biopsies hours At 65 C fixation occurs in - 2 hours II. Alcohol (Ethyl Alcohol) Absolute alcohol alone has very little place in routine fixation for histopathology. It acts as a reducing agents, become oxidized to acetaldehyde and then to acetic acid. 9

10 It is slow to penetrate, hardens and shrinks the tissue. Alcohol penetrates rapidly in presence of other fixative hence in combination e.g. Carnoy's fixative is used to increase the speed of tissue processing. Ethanol preserves some proteins in relatively undenatured state so that it can be used for immunofluorescence or some histochemical methods to detect certain enzymes. It is a fat solvent hence it dissolve fats and lipids Methyl alcohol is used for fixing blood and bone marrow smears. III. Acetone : Cold acetone is sometimes used as a fixative for the histochemical demonstration of some tissue enzymes like phosphatases and lipases. Its mode of action as fixative is similar to that of alcohol IV. Mercuric Chloride (HgCl 2 ) Mercuric chloride is a very good salt employed in fixing but is rarely used alone because it causes shrinkage of the tissue. It brings about precipitation of the proteins which are required to be removed before staining by using potassium iodide in which they are soluble. The size (thickness) of the tissue to be fixed in mercuric chloride is important, since if the tissue is more than 4 mm, then it hardens the tissue at the periphery whereas the centre remains soft & under fixed. 10

11 It penetrates rapidly without destroying lipids. It neither fixes nor destroys carbohydrates. Treatment of the tissue with mercuric chloride brings out more brilliant staining with most of the dyes. Tissues fixed with mercuric chloride containing fixatives contain black precipitates of mercury which are removed by treating with 0.5% iodide solution in 70% ethanol for 5-10 minutes, sections are rinsed in water, decolourized for 5 minutes in 5% sodium thiosulphate and washed in running water. V. Picric acid - It produces marked cells shrinkage hence it is not used alone. It has to be stored in a damp place because of its explosive nature it is preferably stored under a layer of water. Advantage It penetrates well and fixes rapidly. It precipitates proteins and combines with them to form picrates some of the picrates are water-soluble so must be treated with alcohol before further processing where the tissue comes into contact with water. Note : All the tissues fixed in picric acid containing fixatives should be thoroughly washed to remove the yellow discolouration to ensure proper staining of tissue sections. If the fixative is not removed by washing thoroughly with time even the embedded tissue looses its staining quality. 11

12 VI. Potassium dichromate It fixes the cytoplasm without precipitation. Valuable in mixtures for the fixation of lipids especially phospholipids. Used for fixing phosphatides and mitochondria. Note - Thorough washing of the tissue fixed in dichromate is required to avoid forming an oxide in alcohol which cannot be removed later. VII. Osimium tetraoxide - It is a strong oxidizing agent and brings about fixation by forming cross links with proteins. It gives excellent preservation of details of a cell, therefore exclusively used for electron microscopy. It fixes fat e.g. myelin. It also demonstrates fat when 0.5-2% aqueous solution is used it gives a black colour to fat. VIII. Acetic acid - It causes the cells to swell hence can never be used alone but should be used with fixatives causing cell shrinkage IX. Glutaradehyde - It is used alone or in combination with osimium tetroxide for electron microscopy. Compound fixatives - Some fixatives are made by combining one or more fixative so that the disadvantage of one are reduced by use of another fixative. All these compound fixative have their own advantages and disadvantages. They should be used judiciously. 12

13 Choice of fixative - The choice of fixative depends on the treatment a tissue is going to receive after fixation e.g. what is the chemical structure that needs to be stained? If fat is to be demonstrated the formalin fixed tissue is better. For demonstration of glycogen formalin should never be used instead alcohol should be the choice of fixative Preparation of the specimen for fixation 1. For achieving good fixation it is important that the fixative penetrates the tissue well hence the tissue section should be > 4mm thick, so that fixation fluid penetrates from the periphery to the centre of the tissue. For fixation of large organs perfusion method is used i.e. fixative is injected through the blood vessels into the organ. For hollow viscera fixative is injected into the cavity e.g. urinary bladder, eyeball etc. 2. Ratio of volume of fixative to the specimen should be 1: Time necessary for fixation is important routinely 10% aqueous formalin at room temperature takes 12 hours to fix the tissue. At higher temperature i.e C the time for fixation is reduced to 2 hours. Fixatives are divided into three main groups A. Microanatomical fixatives - such fixatives preserves the anatomy of the tissue. 13

14 B. Cytological fixatives - such fixation are used to preserve intracellular structures or inclusion. C. Histochemical fixatives : Fixative used to preserve he chemical nature of the tissue for it to be demonstrated further. Freeze drying technique is best suited for this purpose. Microanatomical fixatives 1. 10% (v/v) formalin in 0.9% sodium chloride (normal saline). This has been the routine fixative of choice for many years, but this has now been replaced by buffered formal or by formal calcium acetate 2. Buffered formation (a) Formalin 10ml (b) Acid sodium phosphate gm (monohydrate) (c) Anhydrous disodium gm phosphate (d) Water to 100 ml - Best overall fixative 3. Formal calcium (Lillie : 1965) (a) (b) (c) Formalin : 10 ml Calcium acetate 2.0 gm Water to 100 ml Specific features - They have a near neutral ph - Formalin pigment (acid formaldehyde haematin) is not formed. 14

15 4. Buffered formal sucrose (Holt and Hicks, 1961) (a) Formalin : 10ml (b) Sucrose : 7.5 gm (c) M/15 phosphate to 100 ml buffer (ph 7.4) Specific features - This is an excellent fixative for the preservation of fine structure phospholipids and some enzymes. - It is recommended for combined cytochemistry and electron microscopic studies. - It should be used cold (4 C) on fresh tissue. 5. Alcoholic formalin Formalin 10 ml 70-95% alcohol 90 ml 6. Acetic alcoholic formalin Formalin Glacial acetic acid Alcohol 70% 5.0ml 5.0 ml 90.0 ml 7. Formalin ammonium bromide Formalin Distilled water Ammonia bromide 15.0 ml 85.0 ml 2.0 gm Specific features : Preservation of neurological tissues especially when gold and silver impregnation is employed 15

16 8. Heidenhain Susa (a) Mercuric chloride 4.5gm (b) Sodium chloride 0.5 gm (c) Trichloroacetic acid 2.0 gm (d) Acetic acid 4.0 ml (e) Distilled water to 100 ml Specific features - Excellent fixative for routine biopsy work - Allows brilliant staining with good cytological detail - Gives rapid and even penetration with minimum shrinkage - Tissue left in its for over 24 hours becomes bleached and excessively hardened. - Tissue should be treated with iodine to remove mercury pigment 9. Zenker's fluid (a) Mercuric chloride 5gm (b) Potassium dichromate 2.5 gm (c) Sodium sulphate 1.0 gm (d) Distilled water to 100 ml (e) Add immediately before use : Glacial acetic acid : 5 ml Specific features - Good routine fixative - Give fairly rapid and even penetration - It is not stable after the addition of acetic acid hence acetic acid (or formalin) should be added just before use 16

17 - Washing of tissue in running water is necessary to remove excess dichromate 10. Zenker formal (Helly's fluid) (a) Mercuric chloride - 5 gm (b) Potassium dichromate 2.5 gm (c) Sodium sulphate 1.0 gm (d) Distilled water to 100 ml (e) Add formalin immediately before use 5 ml Specific features - It is excellent microanatomical fixative - Excellent fixative for bone marrow spleen and blood containing organs - As with Zenker's fluid it is necessary to remove excess dichromate and mercuric pigment 11. B5 stock solution Mercuric chloride Sodium acetate Distilled water 12 gm 2.5gm 200ml B5 Working solution B5 stock solution 20ml Formalin (40% w/v formaldehyde) 2 ml Specific Features - B5 is widely advocated for fixation of lymphnode biopsies both to improve the cytological details and to enhance immunoreactivity with 17

18 antiimmunoglobulin antiserum used in phenotyping of B cell neoplasm. Procedure Prepare working solution just before use Fix small pieces of tissue (7x7x2.5mm) for 1-6 hours at room temperature Process routinely to paraffin. 12. Bouin's fluid (a) Saturated aqueous picric acid 75ml (b) Formalin 25ml (c) Glacial acetic acid 5 ml Specific features - Penetrates rapidly and evenly and causes little shrinkage - Excellent fixative for testicular and intestinal biopsies because it gives very good nuclear details, in testes is used for oligospermia and infertility studies - Good fixative for glycogen - It is necessary to remove excess picric acid by alcohol treatment 13. Gender's fluid - better fixative for glycogen. (a) Saturated picric acid in 95% v/v/ alcohol 80ml (b) Formalin 15ml (c) Glacial acetic acid 5ml 18

19 Cytological fixatives Subdivided into (A) (B) Nuclear fixatives Cytoplasmic fixatives A. Nuclear fixatives : As the name suggests it gives good nuclear fixation. This group includes 1. Carnoy's fluid. (a) Absolute alcohol 60ml (b) Chloroform 30ml (c) Glacial acetic acid 10 ml Specific features - It penetrates very rapidly and gives excellent nuclear fixation. - Good fixative for carbohydrates. - Nissil substance and glycogen are preserved. - It causes considerable shrinkage. - It dissolves most of the cytoplasmic elements. Fixation is usually complete in 1-2 hours. For small pieces 2-3 mm thick only 15 minutes in needed for fixation. 2. Clarke's fluid (a) (b) Absolute alcohol 75 ml Glacial acetic acid 25 ml. 19

20 Specific features - Rapid, good nuclear fixation and good preservation of cytoplasmic elements. - It in excellent for smear or cover slip preparation of cell cultures or chromosomal analysis. 3. New Comer's fluid. (a) Isopropranolol 60 ml (b) Propionic acid 40ml (c) Petroleum ether 10 ml. (d) Acetone 10 ml. (e) Dioxane 10 ml. Specific features - Devised for fixation of chromosomes - It fixes and preserves mucopolysacharides. Fixation in complete in hours. (b) Cytoplasmic Fixatives (1) Champy's fluid (a) 3g/dl Potassium dichromate 7ml. (b) 1% (V/V) chromic acid 7 ml. (c) 2gm/dl osmium tetraoxide 4 ml. Specific features - This fixative cannot be kept hence prepared fresh. - It preserves the mitochondrial fat and lipids. 20

21 - Penetration is poor and uneven. - Tissue must be washed overnight after fixation. (2) Formal saline and formal Calcium Fixation in formal saline followed by postchromatization gives good cytoplasmic fixation. Histochemical fixatives For a most of the histochemical methods. It is best to use cryostat. Sections are rapidly frozen or freeze dried. Usually such sections are used unfixed but if delay is inevitable then vapour fixatives are used. Vapour fixatives 1. Formaldehyde- Vapour is obtained by heating paraformaldehyde at temperature between 50 and 80 C. Blocks of tissue require 3-5 hours whereas section require ½- 1 hours. 2. Acetaldehyde- Vapour at 80 C for 1-4 hours. 3. Glutaraldehyde- 50% aqueous solution at 80 C for 2 min to 4 hours. 4. Acrolein /chromyl chloride- used at 37 C for 1-2 hours Other more commonly used fixatives are (1) formal saline (2) Cold acetone Immersing in acetone at 0-4 C is widely used for fixation of tissues intended to study enzymes esp. phosphates. (3) Absolute alcohol for 24 hours. Secondary fixation - Following fixation in formalin it is sometimes useful to submit the tissue to second fixative eg. mercuric chloride for 4 hours. It provided firmer texture to the tissues and gives brilliance to the staining. 21

22 Post chromation- It is the treatment and tissues with 3% potassium dichromate following normal fixation. Post chromatization is carried out either before processing, when tissue is for left for 6-8 days in dichromate solution or after processing when the sections are immersed in dichromate solution, In for hours, in both the states washing well in running water is essential. This technique is used a mordant to tissues. Washing out- After the use of certain fixative it in urgent that the tissues be thoroughly washed in running water to remove the fixative entirely. Washing should be carried out ideally for 24 hours. Tissues treated with potassium dichromate, osimium tetraoxide and picric acid particularly need to be washed thoroughly with water prior to treatment with alcohol (for dehydration). Tissue Fixative of choice Time for fixative Routine Formalin hours. GIT biopsies buffered formaldehyde 4-6 hours Testicular biopsy Bouin's fixative 4-6 Hours. Liver Biopsy Buffered formaldehyde 4-12 hours. Bone marrow biopsy Bouin's fixative in running 2½ hours followed by washing in running water overnight 1-6 hours Spleen and blood filled Zenker's fluid cavities Lymph node B hours Mictocondria, Carnoy's fluid 1-2 hours phosphatides and Nissil substance Chromosome / cell culture Clarke's fluid 1-2 hours 22

23 CHAPTER 3 DECALCIFICATION Specific Objective - The aim of the study is to ensure staining of hard bony lesions so that the study of pathological lesions is possible. Mode of teaching - Overhead projector and practical demonstration. Definition Decalcification is a process of complete removal of calcium salt from the tissues like bone and teeth and other calcified tissues following fixation. Decalcification is done to assure that the specimen is soft enough to allow cutting with the microtome knife. Unless the tissues in completely decalcified the sections will be torn and ragged and may damage the cutting edge of microtome knife. The steps of decalcification 1. To ensure adequate fixation and complete removal of the calcium it is important that the slices are 4-5 mm thick. Calcified tissue needs 2-3 hours only, for complete decalcification to be achieved so it in necessary to check the decalcification after 2-3 hours. 2. Fixative of choice for bone or bone marrow is Zenker formal or Bouin's fluid. Unfixed tissue tends be damaged 4 times greater during decalcification than a properly fixed tissue. Decalcification Decalcification is effected by one of the following methods. (a) Dissolution of calcium by a dilute mineral acid. 23

24 (b) Removal of calcium by used of dilute mineral and along with ion exchange resin to keep the decalcifying fluid free of calcium. (c) (d) Using Chelating agents EDTA. Electrolytic removal of calcium ions from tissue by use of electric current. The Criteria of a good decalcifying agents area. 1. Complete removal of calcium. 2. Absence of damage to tissue cells or fibres. 3. Subsequent staining not altered. 4. Short time required for decalcification. Removal of calcium by mineral acids - Acid decalcifies subdivided into- Strong acid, weak acid. Strong acid - eg. Nitric and hydrochloric acid. Nitric acid- 5-10% aqueous solution used. They decalcify vary rapidly but if used for longer than hrs. cause deterioration of stainability specially of the nucleus Hydrochloric acid % aqueous solution decalcification slower than nitric acid but still rapid. Fairly good nuclear staining. Weak acid e.g. formic, acetic and picric acid of these formic acids is extensively used as acid decalcifier. 5-10% aqueous solution or with additives like formalin or buffer are used. 24

25 Formic acid 1. Brings out fairly rapid decalcification. 2. Nuclear staining in better. 3. But requires neutralization and thorough washing prior to dehydration. Aqueous nitric acid Nitric acid Distilled water 5-10 ml to 100 ml. Procedure 1. Place calcified specimen in large quantities of nitric acid solution until decalcification is complete (change solution daily for best results). 2. Washing running water for 30 minutes 3. Neutralize for a period of at least 5 hours in 10% formalin to which excess of calcium or magnesium carbonate has been added. 4. Wash in running water over night 5. Dehydrate, clear and impregnate in paraffin or process as desired. Note: Overexposure to nitric acid impairs nuclear staining. Nitric acid is the solution of choice for decalcifying temporal bones. Perenyi's fluid 10% nitric acid 40.0ml Absolute alcohol 30.0 ml. 0.5% chromic acid ml. Note all these ingredients may be kept in stock and should be mixed immediately before use. This solution may acquire of blue violet tinge after a short while but this will have no effect in the decalcifying property. 25

26 It is slow for decalcifying hard bone but excellent fluid for small deposits of calcium eg. calcified arteries, coin lesions and calcified glands. Also good for human globe which contains calcium due to pathological conditions. There is little hardening of tissue but excellent morphologic detail is preserved. Formalin Nitric acid Formalin Distilled water Nitric acid 10 ml 80 ml 10ml Nitric acid causes serious deterioration of nuclear stainability which partially inhibited by formaldehyde. Old nitric acid also tends to develop yellow discolouration which may be prevented by stabilization with 1% urea. Aqueous formic acid 90% formic acid 5-10 ml Distilled water to 100 ml. Gooding and Stelwart's fluid. 90% formic acid 5-10ml. Formalin Distilled water 5ml to 100 ml. Evans and Krajian fluid 20% aqueous trisodium citrate 65 ml 90% formic acid 35 ml This solution has a ph of

27 Formic acid sodium citrate method Procedure 1. Place calcified specimen in large quantities of formic acid-sodium citrate solution until decalcification is complete (change solution daily for best results). 2. Wash in running water for 4-8 hours 3. Dehydrate, clear and impregnate with paraffin or process as desired. This technique gives better staining results then nitric acid method, since formic acid and sodium citrate are less harsh on the cellular properties. Therefore even with over exposure of tissue in this solution after decalcification has been complete, causes little loss of staining qualities. This method of choice for all orbital decalcification including the globe. Surface decalcification- The surface of the block to be decalcified is trimmed with scalpel. The block is then placed in acid solution at 1% hydrochloric acid face downwards so that acid bathes the cut surface for min. As penetration and decalcification is only sufficient for a few sections be cut the block shall be carefully oriented in microtome to avoid wastage of decalcified tissue. Decalcification of Bone marrow biopsy. Tissue after fixation in Bouin's or Zenker's fixative is decalcified for 2½ hours followed by an hour of washing. The tissue in then dehydrated beginning with alcohol. 27

28 Use of Ion exchange resins Ion exchange resins in decalcifying fluids are used to remove calcium ion from the fluid. Therefore ensuring a rapid rate of solubility of calcium from tissue and reduction in time of decalcification. The resins an ammoniated salt of sulfonated resin along with various concentrations of formic acid are used. The resin in layered on the bottom of a container to a depth of = ½ inch, the specimen is allowed to rest in it. After use, the resin may be regenerated by washing twice with dilute N/10 HCL followed by three washes in distilled water. Use of Ion exchange resin has advantage of (ii) faster decalcification (ii) tissue preservation and (iii) cellular details better preserved. Chelating agents Chelating agents are organic compounds which have the power of binding certain metals. Ethylene-diamene-tetra-aceticacid, disodium salt called Versenate has the power of capturing metallic ions. This is a slow process but has little or no effect on other tissue elements. Some enzymes are still active after EDTA decalcification. Versenate Distilled water 10 gm. 100 ml (ph 5.5 to 6.5) Time 7-21 days. 28

29 Electrolytic method This is based on the principle of attracting calcium ions to a negative electrode in to addition to the solution. Decalcifying solution HCL (Conc.) 80ml Formic acid 90% 100 ml Distilled water 1000 ml. Decalcify with electrolyte apparatus with the above mentioned decalcifying fluid. This method has no added advantage over any other method. Neutralization : It has been said that following immersion in mineral acids, tissues should be deacidified or neutralized, before washing by treatment with alkali. This may be effected by treatment over night in 5% lithium or sodium sulphate. Washing : Through washing of the tissue before processing is essential to remove acid (or alkali if neutralized has been carried out) which would otherwise interfere with staining) Determination of end point of decalcification 1. Flexibility method Bending, needling or by use of scalpel if it bends easily that means decalcification is complete. Unreliable, causes damage and distortion of tissue. 29

30 2. X-ray method Best method for determining complete decalcification but very costly. Tissue fixed in mercuric chloride containing fixatives cannot be tested as they will be radio opaque. 3. Chemical Method It is done to detect calcium in the decalcifying fluid when no further calcium is detected, decalcification in considered complete. Procedure Take 5 ml of decalcifying fluid from the bottom of container which has been in contact with the tissue for 6-12 hrs. Add 5 ml each of 5% ammonium oxalate and 5% ammonium hydroxide. Mix and let it stand for min. A cloudy solution caused by calcium oxalate indicates that specimen is not thoroughly decalcified. Absence of turbidity indicates completeness of decalcification. Treatment of hard tissues Keratin and chitin are softened by use of concentrated sulphuric and with that aid of heat keratin is completely dissolved from the tissue sections. But much tissue distortion will also occur. For softening of chitin foll procedure gives a satisfactory result. 1. Fix the specimen in fixative of choice. 2. Place the specimen in following solution until complete dechitinized. Change the solution every two days for best results. 30

31 Mercuric chloride - 4 gm Chromic acid - 0.5gm Nitric acid (Conc.) - Ethyl alcohol 95% ml 50.0 ml Distilled water ml 3. Washing running water for 3 hours 4. Dehydrate, clear and impregnate with paraffin. Prenyi's fluid Immersing hard tissues in this solutions for hours will make sectioning easier and excellent preparation of calcified arteries, thyroid and calcified glands is possible. Lendrum's technique It is very useful for tissues which became hard at the time of fixation. Following washing out of the fixative, tissue is immersed in a 4% aqueous solution of phenol for 1-3 days. Wax blocks - The treatment of wax embedded block of hard tissue may be done by soaking in soap water overnight. 31

32 CHAPTER 4 TISSUE PROCESSING Specific objective - The tissue processing is the heart of any tissue section which will be cut adequately only if the tissue is properly preserved and processed. The study of this topic is to understand the coarse and fine details of tissue processing so that excellent sections are obtained. Mode of teaching - Overhead projector, slide projector and practical demonstration. Definition - The term tissue processing refers to treatment of the tissue necessary to impregnate it into a solid medium so that the tissue is rendered sufficiently firm yet elastic for the tissue sections of desirable thickness to be cut on microtome. This is not the only technique employed for tissue sections. Sections can also be produced by means of crytostat or freezing microtome on frozen tissues. The fixed impregnated tissues have an advantage that they can be more easily stored and reproducibility of sections at a later date is easier. Before proceeding on tissue processing as soon as the tissue is received it in very important that the tissue be properly labeled so as to avoid any confusion regarding duplication of same name or giving a wrong diagnosis to the patient. The labeling has to be a full proof system. The label should remain throughout the entire processing and later as 32

33 permanent record keeping. To ensure this most laboratories have a numbering system for each specimen. As soon as the specimen is received it is given a specific individual number, which is also recorded in the register with the details like patient's name, name of the doctor referring it, nature of tissue is noted. Labeling should not be done using ordinary ink as it gets dissolved in the reagent used during processing. Thin white card with a soft lead pencil, typed or printed labels are satisfactory. To ensure that the label remains with their correct specimens tissues processing baskets can be used. These are small perforated metal containers in which the tissue and labels are placed. these containers can be transferred as such from reagent to reagent. Alternatively use of tissue tek system in which the tissue identity is written on the cassette and retained as permanent record during sectioning and storage of tissue blocks. Principle of tissue processing - The tissue is embedded in a solid medium by the help of first removing the tissue water which is then replaced by any solid medium such as paraffin wax so that the tissue is rendered firm enough to enable thin sections to be cut, at the same time, the tissue is soft (not so hard) to enable microtome knife to cut the sections. The embedding medium has to thoroughly permeate the tissue in fluid form so that it solidifies without any damage to the tissue. The most satisfactory embedding medium used in routine histology is paraffin wax. Most of the tissue fixatives are aqueous fixatives so before the tissue can be 33

34 embedded in paraffin wax it is necessary that the water and some of the lipid tissue fluids be removed completely by a variety of compounds through a process called dehydration. Prior to paraffin wax embedding and impregnation the tissue must be subjected to the following steps: 1. Fixation 2. Dehydration - 3. Clearing - with a substance which is totally miscible with both the dehydrating agent which precedes it, and embedding agent which follows it. 4. Embedding All these 4 processes depend upon complete impregnation of the tissue by the agent like paraffin wax being used. Before going into the details of these 4 stages it is important to understand the factors which influence the rate and efficiency of tissue impregnation Factors influencing the rate of impregnation A tissue immersed in fluid interchange occurs between tissue fluid and surrounding fluid. The process continues through all stages of processing from fixation to final impregnation. Agitation - Tissue placed in liquid is agitated so that the fluid immediately in contact with the surface of tissue which is mixed by tissue fluid is replaced by the fresh immersing liquid. 34

35 This can be achieved by a pumping system which removes and replaces fluid at selected intervals or by rotation and vertical oscillation method. Efficient agitation reduces the processing time by 25-30% with improved impregnation of the tissue. Heat - Heat increases the rate of penetration. Viscosity - Larger the molecule the higher is the viscosity slower is the rate of penetration. Ultrasonic : Use of ultrasonics increases the penetration rate. Vacuum : Use of reduced pressure in well known in the impregnation of tissue by molten paraffin wax. It hastens the process. Use of vacuum during dehydration and clearing has little advantage except removal of air bubble trapped within the tissue. STEPS OF PARAFFIN WAX EMBEDDING Fixation - Usually tissue that is received at the laboratory is already fixed but before proceeding further check if the fixation is complete. Dehydration - After fixation in aqueous solvent the delicate tissue needs to be dehydrated slowly starting in 50% ethyl alcohol. The other routine tissue specimen may be put in 70% alcohol. A higher concentration of alcohol initially is in inadvisable because this may cause very rapid removal of water may produce cell shrinkage. An exception to this is in case of Heidenhain's Susa fixed tissue where it may be placed directly in 95% alcohol. Tissue transferred from alcoholic based fixative like Carnoy's fixative may be placed in higher grades of alcohol or even in absolute alcohol. 35

36 For routine biopsy and postmortem tissue of 4-7 mm thickness 70%, 90% and absolute alcohol (2-3 changes for 2-4 hours each) are sufficient to give reasonably satisfactory result. Use of solid dehydrants Anhydrous copper sulphate is used in higher grade of dehydrating alcohols. A layer cm thick is placed at the bottom of a dehydrating vessel or beaker and is covered with 2 or 3 filter papers to prevent contamination of the tissues. Anhydrous copper sulphate is white, it removes water from alcohol which in turn has been diluted upon absorption of water from the tissues. The change of colour of copper sulphate from white to blue indicates that both alcohol and water should be changed. Use of copper sulphate enhances the process of dehydration and also prolongs the life of alcohol. Other dehydrating agents 1. Acetone - It is clear, colourless volatile inflammable fluid. It has a rapid action in dehydrating the tissue but produces shrinkage and distortion and subsequent brittleness to the tissue. Low cost is also an advantage. Acetone usually dehydrates within minutes but four changes of acetone should be used, it is preferable to use acetone after low strength of alcohol so that distortion of the tissue is less. 36

37 2. Dioxane - It dehydrates and clears at the same time. It is miscible with paraffin and with water and alcohol, tissue from dioxane can be transferred straight to paraffin. There is less shrinkage of tissues Tissues can be left in dioxane without danger of hardening for longer period of time. Disadvantage: It is more expensive than alcohol. * It is toxic to man 3. Isopropyl alcohol It is miscible with water and other organic solvents It does not harden the tissue like alcohol It is expensive Clearing Definition - Clearing means appearance of tissue after it has been treated by the fluid chosen to remove the dehydrating agent. Most of these tissues have similar refractive index to that of protein therefore the tissue is left translucent. Clearing agent is required when the dehydrating agent is not miscible with the impregnating medium. It is essential for a clearing agent to be miscible both in dehydrating agent as well as embedding agent. 37

38 Commonly used clearing agents are as follows : 1. Xylene - It has a rapid action. Biopsy specimens of 3-4 mm thickness are cleared in 2-4 hours. Immersion time must not be prolonged otherwise the tissue become brittle. 2. Toluene and Benzene are similar in properties to xylene but are less damaging to the tissues on prolonged exposure. 3. Chloroform - It is slower in action but it causes less brittleness therefore tissue can be left in it overnight. It does not affect the refractive index of the tissue is not rendered translucent. It is expensive. It is inflammable. 4. Carbon tetrachloride - It has similar properties to chloroform but is cheaper. 5. Cedar wood oil (Histological): It is good for treatment of delicate tissues as it has the least hardening effect. It is very slow in action. It is very expensive. Care should be taken not to confuse it with cedar wood oil (microscopic) used with oil immersion lens. Techniques of clearing : If the tissue is being cleared in chloroform or carbon tetrachloride it may be left overnight. In automatic tissue processor three changes of one hour each are usually satisfactory. 38

39 In Xylene, benzene or toluene one change after minutes is satisfactory to give a clear translucent appearance to the tissue. Impregnation Definition - It is the complete removal of clearing reagents by substitution of paraffin or any such similar media. Impregnation with wax Impregnation with paraffin wax takes place in an oven heated to C depending upon the melting point of the wax in use. Frequent check of the temperature of paraffin baths is required since temperature 5 C above the melting point of the paraffin will cause tissue shrinkage and hardening. Properties of paraffin wax 1. Easy to prepare large number of tissue blocks in comparatively short time. 2. Minimum supervision is required 3. It is cheaper than other impregnating media 4. During staining there is very little difficulty than other media. Points to be remembered during use of paraffin wax 1. It should be free from dust, grit and other foreign matter. 2. It should not contain water, which causes it to crystallize and turn it white. 39

40 3. The wax has to be filtered before use by use of ordinary filter paper. 4. Higher melting point waxes are hard to ribbon. For impregnation the wax oven has to be kept at high temperature, making the tissue hard, too low melting point wax may not be hard enough to support the tissue during cutting. If the wax is overheated and remains in that state for a long time, it tends to crystallize and become useless. Paraplast - This is mixture of highly purified paraffin and several plastic polymers. It has greater elasticity than normal paraffin wax, therefore, the results are superior. It ribbons well allowing almost wrinkle free serial sections to be cut with ease at 4 micron thickness. It should not be used for thin walled structures as it prevents complete expansion of the specimen. Bioloid - Good embedding medium in which thin walled structures can be sectioned satisfactorily. Technique of impregnation : The tissue is transferred from clearing agent to molten paraffin wax. The amount of wax should be times the volume of tissue. The tissue must be submitted to 3 changes in wax. The temperature of the wax bath should be 2-3 C above the melting point of wax. 40

41 Time of impregnation Depends on the following 3 factors 1. The size and type of tissue 2. The clearing agent employed 3. The use of vacuum embedding oven. 1. Size and type of tissue: The thicker the tissue the longer will be the time required for wax to penetrate to the centre in addition a thick tissue has more of clearing agent so more changes of wax are necessary to remove it. If even small amounts of clearing agents remains with the wax this will cause crystallization and produce crumbling of the sections during cutting. The type of tissue is also important since bone, skin, CNS needs twice as long as soft tissue like liver or kindly. Tissue like muscle and fibrous tissue tends to overharden and become brittle in wax bath so the time for impregnation must be kept to a minimum. The reduction of time can be achieved by using vacuum embedding medium. 2. Clearing agent employed Some clearing agents are more rapidly and easily cleared than other e.g. Xylene, benzene and toluene are easiest to remove, and one change of wax is normally sufficient; whereas for chloroform and carbon tetrachloride 2-3 changes are needed. 41

42 3. Use of vacuum embedding oven With the use of normal paraffin oven, 2 changes of paraffin wax for a period of 4 hours are needed but by using vacuum embedding oven this time may be halved Embedding - It is the orientation of tissue in melted paraffin which when solidified provides a firm medium for keeping intact all parts of the tissue when sections are cut. Types of moulds a) Leuckhart's L pieces - These are two 'L' which are resting metal usually brass, which are resting on a flat metal or glass plate. 1. L-moulds or embedding box b) Compound embedding units - consists of square shaped brass or metal plates in a series of interlocking plates. c) Others like plastic embedding blocks (tissue Tek system) Techniques of casting 1. Molten paraffin wax which is heated at a temperature 2-3 above the melting point is poured into the mould to an adequate depth so as to cover the thickest tissue block. 42

43 2. The wax touching the mould will quickly form a thin semi solid layers, Now introduce the tissue with a prewarmed forceps to prevent the wax to stick to it. The tissue is pressed in this semisolid wax to orient it at the bottom of mould in a correct plane. 3. Fix the label in position by pressing one edge against solidifying wax usually sides of the mould are preferred. 4. As soon as a film of solid wax is formed on the surface, the whole block with mould are submerged in cold water at 20 C. If this is not done there will be crystallization of wax, using ice water to do initial cooling will also cause the block to crack. 5. When blocks are set hard they are removed from mould. The tissue surface towards the mould base is from where the sections are to be cut this surface should be trimmed lightly with a scalpel so as to expose the tissue. Following points must be taken care off during casting. 1. Paraffin should not be allowed to cool around the tissue to be blocked for this before introducing the tissue in the mould it should be kept in heated wax or in cassette placed over thermostatic hot plate. 2. To prevent excess of wax solidifying on the bottom of the block during winter prewarmed moulds may be used. 3. The cutting surface of the tissue should be facing at the bottom of the mould. 43

44 4. If 2 or more tissues have to be casted remember to keep them both at the same depth. 5. If small biopsy fragments have to be casted, the largest piece should be first blocked and other pieces should be as near it as possible. 6. All four corners of the block should be in one horizontal plane. 7. The tissue should have atleast 2 mm wax around its edges. 8. Smear mineral or machine oil on the inner surface of the mould for facilitating easy removal of block. 9. Whitish areas around tissue in block denotes crystalization which may be due to moisture or due to incomplete removal of clearing agent. Most tissue sections are cut from the largest area but some tissue needs special mention. 1. Tissue of tubular nature are cut transversely so should be embedded vertically. 2. Skin is cut in a plane at right angles to the surface so should be embedded at right angles to the bottom. 3. Muscle biopsy should be sectioned in both transverse and longitudinal planes. Automatic tissue processor It has 2 advantages 1. Transferring the tissue mechanically from one reagent to another can be done both by day and night. 44

45 2. Reduces processing time by the action of continuous agitation. 3. This eliminates the possibility of human errors of leaving the tissue for long time in one solution due to forgetfulness. Paraffin embedding (2-4) The process of tissue embedding in L-mould (5) Plastic mould and (6) Paraffin block VARIOUS PARTS OF THE MACHINE ARE AS FOLLOWS (a) Tissue containers - These are also the cassettes. The tissue to be processed is placed in an appropriate container, together with a label and the lid snapped on. These containers are placed in the tissue basket in which they remain throughout the whole process. (b) Beakers and wax baths - Most machines are equipped with ten beakers and 2 wax baths thermostatically controlled at 56 C + 4 C. The beakers are filled with appropriate fluids and wax is placed in the wax baths after ensuring that main switch is on, so as to keep the wax in molten state. 45

46 (c) Stirring mechanism - The basket is attached to the arms of the machine on which one arm is designed in such a manner so as to bring about the rotation of the basket nearly at the rate of one revolution per minute. (d) Timing mechanism - Timer is meant to keep the tissue in different reagents and wax for an optimum time. If kept for longer or shorter period than necessary, tissue will not be adequately processed. Points to noted 1. Fluid and wax beakers must be filled upto appropriate mark and located in their correct position in the machine. 2. Any spillage of the fluid should be wiped away. 3. Accumulations of wax must be removed from beaker, covers, lids and surrounding areas. 4. Wax bath thermostats should be set at satisfactory levels usually 2-3 C above the melting point of wax. 5. Particular attention should be paid to fastening the processing baskets on the crousel type of machines, if the baskets are shed they will remain in one particular regent for a long period till it gets noticed. 6. Timing should be set with utmost care when loading the machine. 7. Paraffin wax baths should be checked to ensure that the wax is molten. 46